Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Proﬁle

cells Article Spermidine and Rapamycin Reveal Distinct Autophagy Flux Response and Cargo Receptor Clearance Profile Sholto de Wet, Andre Du Toit and Ben Loos * Department of Physiological Sciences, Stellenbosch University, Stellenbosch 7600, South Africa; [email protected] (S.d.W.); [email protected] (A.D.T.) * Correspondence: [email protected]; Tel.: +27-21-808-9196; Fax: +27-21-808-3145 Abstract: Autophagy flux is the rate at which cytoplasmic components are degraded through the entire autophagy pathway and is often measured by monitoring the clearance rate of autophagosomes. The specific means by which autophagy targets specific cargo has recently gained major attention due to the role of autophagy in human pathologies, where specific proteinaceous cargo is insufficiently recruited to the autophagosome compartment, albeit functional autophagy activity. In this context, the dynamic interplay between receptor proteins such as p62/Sequestosome-1 and neighbour of BRCA1 gene 1 (NBR1) has gained attention. However, the extent of receptor protein recruitment and subsequent clearance alongside autophagosomes under different autophagy activities remains unclear. Here, we dissect the concentration-dependent and temporal impact of rapamycin and spermidine exposure on receptor recruitment, clearance and autophagosome turnover over time, employing micropatterning. Our results reveal a distinct autophagy activity response profile, where the extent of autophagosome and receptor co-localisation does not involve the total pool of either entities and does not operate in similar fashion. These results suggest that autophagosome turnover and specific cargo clearance are distinct entities with inherent properties, distinctively contributing towards total functional autophagy activity. These findings are of significance for future studies where disease specific protein aggregates require clearance to preserve cellular proteostasis and viability and highlight the need of discerning and better tuning autophagy machinery activity and Citation: de Wet, S.; Du Toit, A.; cargo clearance. Loos, B. Spermidine and Rapamycin Reveal Distinct Autophagy Flux Keywords: autophagy; autophagy flux; cargo receptor; co-localisation; recruitment; turnover Response and Cargo Receptor Clearance Profile. Cells 2021, 10, 95. https://doi.org/10.3390/ cells10010095 1. Introduction Received: 3 December 2020 Macroautophagy is a major intracellular degradation pathway critical in protein re- Accepted: 30 December 2020 moval and the maintenance of cellular homeostasis [1]. Its dysfunction has been associated Published: 7 January 2021 with the onset of neurodegenerative diseases, typically characterised by the presence of distinct protein inclusion bodies within brain regions associated with the disease [2–4]. Publisher’s Note: MDPI stays neu- Indeed, the overall abundance of autophagosomes, the functional unit of autophagy, is tral with regard to jurisdictional clai- increased in neurons during the pathogenesis of Alzheimer’s as well as Parkinson’s dis- ms in published maps and institutio- ease [5,6]. Moreover, lysosomal storage diseases, such as Gaucher disease [7] or Pompe nal affiliations. disease [8] have been characterized by autophagy dysfunction and selective cargo aggre- gation, with the former presenting a major risk factor for the onset of Parkinson’s disease. Increasing autophagy activity has been shown to aid in the clearance of toxic proteinaceous cargo [9–11], therefore, the precision control of autophagy activity and subsequent Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. enhanced removal of particular cargo has been gaining increasing attention [12–15]. This article is an open access article Three variants of autophagy exist, of which macroautophagy is the most charac- distributed under the terms and con- terised and best explored. Macroautophagy, hereafter referred to as autophagy, is primarily ditions of the Creative Commons At- activated during starvation conditions and is particularly involved in cytoplasmic bulk tribution (CC BY) license (https:// degradation targeting mainly long-lived proteins. In doing so, this process generates creativecommons.org/licenses/by/ metabolic substrates and maintains the cell’s energetic state [16,17]. Basal autophagy activ- 4.0/). ity varies according to cell type [18] and serves a “house-keeping” function; eliminating Cells 2021, 10, 95. https://doi.org/10.3390/cells10010095 https://www.mdpi.com/journal/cells Cells 2021, 10, 95 2 of 19 old or damaged cellular components that may lead to the disruption of homeostasis [4]. Furthermore, the expression profile of microtubule-associated protein light chain 3 (LC3), a critical protein recruited to the autophagosome, has been shown to vary substantially between tissue types, supporting the notion of distinct, tissue-specific autophagy activity [18,19]. However, the number of autophagosomes per cell may rise either due to increased autophagosome synthesis, brought about by pharmaceutical induction for example, or due to their accumulation as a consequence of disrupted cellular homeostasis, brought about by proteotoxicity or lysosomal dysfunction [20,21]. Determining the autophagy activity, or autophagy flux, which is defined as the rate at which material is degraded through the entire autophagy pathway [22], has hence received major attention [15,23,24], particularly since increased autophagy activity has been shown to minimise the harmful effects that arise in the pathogenesis associated with neurodegenerative diseases [25–28]. Autophagy is typically characterised as a sequential process which is initiated by the synthesis of the pre-autophagosome structure; the phagophore, which matures into an autophagosome [29,30]. These will subsequently sequester cytoplasmic components, in- cluding proteinaceous cargo, and be delivered to hydrolase-containing lysosomes where degradation takes place [1]. Importantly, recent evidence supports the notion of a careful discernment between the clearance rate of proteins, as autophagy cargo, contrasted by the autophagosome turnover, to better dissect the efficiency and capacity of the cell to clear proteinaceous components [31,32]. Moreover, it is becoming increasingly clear that autophagy is crucial in alleviating cellular stress through the selective degradation of specific cytoplasmic components [33]. In fact, a plethora of complex and dynamically interactive “receptor” proteins exist and engages with the autophagosome machinery, facilitating cargo-specific degradation [34–36]. These proteins include domains that allow binding to ubiquitinated cytoplasmic components with LC3-II, thereby enabling the targeting of ubiquitinated proteinaceous cargo to the autophagosome [37]. In this manner, the autophagy system responds selectively by recruiting autophagy receptors and hence, is well equipped to degrade specific cargo targets [13]. A key protein of interest, p62/sequestosome-1, is fundamentally engaged during autophagy progression and is largely used as an additional molecular indicator of autophagy activity, along with LC3-II [26,38,39]. Of note, p62 has been found to be the primary receptor protein involved in aggrephagy, the autophagy- dependent degradation of proteins [34]. Additionally, NBR1 has been described in the degradation of protein aggregates. Indeed, major cross talk between p62 and NBR1 has been revealed, with NBR1 levels increasing upon p62 knockout [35], suggesting a complex interplay between receptor recruitment, cargo clearance and autophagy activity. Given the crucial role of autophagy activity in controlling neuronal fate through enhanced clearance of particular protein cargo, this relationship deserves urgent attention. The nature of the relationship between autophagosome turnover and receptor clearance is currently largely unclear. It is also unknown to what extent mTOR-dependent or -independent autophagy induction may govern such change in the autophagosome pool, autophagosome turnover and receptor clearance. In light of these complexities, we aimed to examine the relationship between autophagosome turnover and selective receptor recruitment and clearance, focusing on the two predominant cargo receptors, p62 and NBR1. By employing rapamycin [40] and spermidine [41] at low and high concentrations, we set out to explore their concentration- and time-dependent effect on autophagosome activity, receptor recruitment and subsequent clearance by taking advantage of a unique single-cell analysis and micropatterning approach [42,43]. 2. Methods and Materials 2.1. Cell Culture and Maintenance Mouse embryonic fibroblast (MEF) cells stably expressing green fluorescent protein (GFP)-LC3 (a kind gift from Noboru Mizushima, Tokyo University, Tokyo, Japan) were utilised and cultured in Dulbecco’s modified Eagle’s medium (DMEM, #41965-039, Life Technologies, Johannesburg, South Africa) supplemented with 10% foetal bovine serum Cells 2021, 10, 95 3 of 19 (#S-0615, Biochrom, Berlin, Germany), 1% antibiotic-antimycotic (#15240-062, Life Tech- nologies); 100 µg/mL streptomycin, 100 U/mL penicillin, and maintained in a humidified ◦ atmosphere with the presence of 5% CO2 at 37 C. MEFs were sub-cultured using trypsin (#25200-072, Life Technologies) to detach adherent cells from flasks. After trypsinisation, DMEM was added in a 2:1 ratio and cells were collected into either a 15 mL (#50015, Biocom Biotech, Centurion, South Africa) or a 50 mL Falcon tube (#50050, Biocom Biotech) depend- ing on the size of the original culture

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